Tag reagent and assay method

ABSTRACT

A reagent comprises: a) an analyte moiety comprising at least two analyte residues, and linked to; b) a tag moiety comprising one or more reporter groups adapted for detection by mass spectrometry, wherein a reporter group designates analyte residue, and the reporter group at each position of the tag moiety is chosen to designate an analyte residue at a defined position of the analyte moiety. A plurality of such reagents, each comprising a different analyte moiety, provides a library of reagents which may be used in assay methods involving a target substance. Analysis of the tag moieties indicates the nature of the analyte moieties bound to the target substance. A method of sequencing nucleic acid employs a library of the reagents to determine the sequence of a target nucleic acid.

This application is a 371 of PCT/GB34/01675 filed on Aug. 1, 1994.

In biological and chemical analyses, the use of analyte moleculeslabelled with reporter groups is routine. This invention addresses theidea of providing reagents having at least two analyte groups linked toone or more reporter groups. Such reagents can be used, in waysdescribed below, to generate much more analytical information than cansimple labelled analytes. It is possible to code reporter groups so thatreagents carrying multiple analyte groups and multiple reporter groupscan by synthesised combinatorially and used simultaneously and thereporter groups resolved in the analytical stage.

WO 93/06121 (Affymax) describes a synthetic oligomer library comprisinga plurality of different members, each member comprising an oligomercomposed of a sequence of monomers linked to one or more identifier tagsidentifying the sequence of monomers in the oligomer. The linkagebetween the oligomer and the identifier tag preferably comprises a solidparticle. The identifier tag is preferably an oligonucleotide.

Proc. Natl. Acad Sci., Vol 89, No. 12, 15 Jun. 1992, pages 5381-5383 (SBrenner and R A Lerner) describe encoded combinatorial chemistry formaking a library of reagents each containing a genetic oligonucleotidetag.

In Rapid Communications in Mass Spectrometry, Vol 6, pages 369-372(1992), G R Parr et al describe matrix-assisted laserdesorption/ionisation mass spectrometry of syntheticoligodeoxyribonucleotides.

In Nucleic Acids Research, Vol 21, No. 15, 25 July 1993, pages3347-3357,E Nordhoff et al describe the ion stability of nucleic acids ininfra-red matrix-assisted laser desorption/ionisation mass spectrometry.to which the analyte moiety and the tag moiety are both attached.Preferably the analyte moiety is a chain of n analyte residues, and thetag moiety is a chain of up to n reporter groups, the reporter group ateach position of the tag chain being chosen to designate the analyteresidue at a corresponding position of the analyte chain. n is aninteger of at least 2, preferably 3 to 20.

The invention may be used for the detection of all analytes of interest.These include, but are not limited to, a protein/peptide chain so thatthe analyte residues are amino acid residues; a nucleicacid/oligonucleotide chain so that the analyte residues are nucleotideresidues; a carbohydrate chain so that the analyte residues are sugarresidues. Additionally the analyte may be a class of small moleculeswith biological, pharmacological or therapeutic activity. For example itcould be a core molecule with the ability to vary various substituentgroups eg. alkyl, esters, amines, ethers etc in a combinatorial mannerwith mass spectrometry tags.

The tag moiety and/or the or each reporter group in it is capable ofbeing observed/detected/analysed so as to provide information about thenature of the analyte moiety, and/or the analyte residues in it.

In one embodiment, the reagent has the formula A--L--R where A is achain of n analyte residues constituting the analyte moiety, L is thelinker, R is a chain of up to n reporter groups constituting the tagmoiety, and n is 2-20, wherein the tag moiety contains informationdefining the location of analyte residues in the analyte moiety.

The tag moiety consists of one or more reporter groups distinguishableby mass and thus capable of being analysed by mass spectrometry. Thereporter groups may be chemically different and thus distinguished fromone another by molecular weight. Or the reporter groups may bechemically identical, but distinguished from one another by containingdifferent isotopes (e.g. ¹² C/¹³ C and ¹ H/² H as discussed below). Thetag moiety is, and/or the reporter groups are, suitable or adapted foranalysis by mass spectrometry e.g. after cleavage by photochemical orother means from the reagent.

The advantages of mass spectrometry as a detection system are: its greatsensitivity--only a few hundred molecules are needed to give a goodsignal; its wide dynamic range and high resolving power--molecules inthe mass range 100 to 200,000 Daltons can be resolved with a resolutionbetter than 0.01; its versatility--molecules of many different chemicalstructures are readily analysed; the potential to image analytes bycombining mass spectrometry with, for example, scanning laserdesorption: and the ability to make quantitative as opposed to merelyqualitative measurements.

Thus mass-labelling combines advantages of radioactivity andfluorescence and has additional attributes which suggest novelapplications.

In another aspect, the invention provides a library of the abovereagents, wherein the library consists of a plurality of reagents eachcomprising a different analyte moiety of n analyte residues. Forexample, the library may consist of 4^(n) reagents each comprising adifferent oligonucleotide chain of n nucleotide residues. The reagentsof the library may be present mixed together in solution.

In another aspect, the invention provides an assay method whichcomprises the steps of: providing a target substance; incubating thetarget substance with the said library of reagents under conditions tocause at least one reagent to bind to the target substance;

removing non-bound reagents; recovering the tag moieties of the or eachbound reagent; and analysing the recovered tag moieties as an indicationof the nature of the analyte moieties bound to the target substance.

The target substance may be immobilised, as this provides a convenientmeans for separating bound from non-bound reagent. In one aspect, thetarget substance may be an organism or tissue or group of cells, and theassay may be performed to screen a family of candidate drugs. In anotheraspect, the target substance may be a nucleic acid, and this aspect isdiscussed in greater detail below.

Reference is directed to the accompanying drawings in which:

FIG. 1 is a general scheme for synthesis of reagents according to theinvention.

FIG. 2 shows reagents with three different systems of tag chainscontaining reporter groups.

FIG. 3a is a diagram showing synthesis of coded oligonucleotides, and

FIG. 3b is a diagram showing reading the code of a tag chain.

FIG. 4 is a diagram showing sequence analysis by progressive ligation.

FIG. 5 is a diagram on extending the sequence read by hybridisation toan oligonucleotide assay.

Legends to FIGS. 1 and 2 are included at the end of this specification.

Reference is directed to the example applications below, describing howthe method may be applied to the analysis of nucleic acid sequences, andto screening candidate drugs.

Synthesis of coded tags

The principle of the method used for tagging multiple analytessimultaneously is similar to that proposed by Brenner and Lerner (1992)for coding peptides with attached nucleic acid sequences. The intentionof their idea is to add a tag which can be amplified by the polymerasechain reaction and read by sequencing the DNA molecule produced.

The structure of reagents is best illustrated by considering how theycould be made. Synthesis starts with a bivalent or multivalent linkerwhich can be extended stepwise in one direction to add a residue to theanalyte and in another to add residue-specific reporter groups (FIG. 1).Suppose we wish to make a mixture of organic compounds, introducingdifferent residues at each stage in the synthesis. For example, themixture could comprise a set of peptides with different amino acidsequences or of oligonucleotides with different base sequences, or a setof variants with potential pharmacological activity with differentgroups attached to a core structure; in each case we wish to label eachstructural variant with a unique tag. This is done by dividing thesynthesis at each step where different residues are added to thecompound of interest, and adding corresponding residues to the tag.

As an example, suppose we wish to make a mixture of 4096hexanucleotides, each with a unique tag. Four samples of a bivalentlinker would be coupled with each of the bases and with the uniquereporter for the base (FIG. 3a). The four samples are then mixed,divided in four and the process repeated. The result is a set ofdinucleotides each with a unique tag. The process is repeated until sixcoupling steps have been completed.

The linker and reporter groups

The linker should have one group that is compatible with analytesynthesis--hydroxyl, amino or sulphydryl group are all suitable forinitiating oligonucleotide synthesis, and similar groups can be found toinitiate other pathways, for example, synthesis of polypeptides. Forsome classes of compounds it may be desirable to start with a "core"compound which forms part of the analyte. The choice of the group(s) forstarting addition of reporters depends on the nature of the reportergroups and the chemistry used to couple them. This chemistry has to becompatible with that used for synthesising the analyte. For the exampleof oligonucleotide synthesis, there are a number of alternatives. Theestablished method uses benzoyl and isopropyl groups to protect thebases, acid-labile trityl groups for temporary protection of the 5'-OHgroups during coupling, and p-cyanoethyl groups to protect thephosphates. The method used for coupling the reporters should not attackthese protecting groups or other bonds in the oligonucleotide, and thesynthesis of the tags should not be affected by the coupling, oxidation,and deprotection used in the extension of the oligonucleotide.

The coupling of the reporter monomers or the capping of the chain, maybe incomplete at each step (FIG. 2, B and C), so that the analyte iscoupled to a nested set of reporter structures. This will make it easierto deduce the structure of the analyte from the composition of the tag(FIG. 1; FIG. 3). To make the synthesis easier it is desirable for thelinker to be attached to a solid support by a linkage which can becleaved without degrading the analyte or the reporter groups.Alternatively, the linker may carry a group such as a charged group or alipophilic group which enables separation of intermediates and the finalproduct from reagents.

The reporter groups could take many forms, the main consideration is theneed to read the composition or sequence of the tag by massspectrometry. Possibilities include groups with different atomic orformula weights, such as aliphatic chains of different lengths ordifferent isotopic composition. Using isotopically labelled methylenegroups, it is possible to assign a group of unique formula weight toeach of four different reporters (Table 1).

                  TABLE 1                                                         ______________________________________                                        Example reporters based on isotopes of hydrogen and carbon:                   Isotopic   Formula Weight                                                     Composition                                                                              (of --OCH.sub.2)                                                                             Symbol  Base                                        ______________________________________                                        .sup.12 CH.sub.2                                                                         30             r.sub.30                                                                              A                                           .sup.12 CHD, .sup.13 CH.sub.2                                                            31             r.sub.31                                                                              C                                           .sup.12 CD.sub.2, .sup.13 CHD                                                            32             r.sub.32                                                                              G                                           .sup.13 CD.sub.2                                                                         33             r.sub.33                                                                              T                                           ______________________________________                                    

Taking the example of oligonucleotides these tags can make a set whichallows the base at each position in the oligonucleotide to be read fromthe incremental masses of the partial products in the series (Table 2).All oligonucleotide sequences will give a unique series of tag fragmentweights provided the smallest increment in adding a reporter is largerthan the mass difference between the smallest and the largest reporter.

                  TABLE 2                                                         ______________________________________                                        Example oligonucleotide with isotopic reporters:                               ##STR1##                                                                     ______________________________________                                    

For mass spectrometry, it will be desirable to have a simple way ofcleaving the tag chain from the analyte. There are severalpossibilities. Among methods compatible with oligonucleotide and peptideanalytes are: light induced cleavage of a photolabile link; enzymaticcleavage, for example of an ester link; free-radical induced cleavage.

A further requirement is that the tags should be compatible with thechemical and biochemical processes used in the analysis: for the exampleof oligonucleotides used in molecular hybridisation or for one of theproposed sequencing methods, they must be soluble and they must notinhibit certain enzymatic reactions which may be used in the analysis.Experience has shown that oligoethylene glycol linkages, similar to themethylene analogues shown in Table 1, are compatible with molecularreassociation of oligonucleotides. Furthermore, such linkages arecompatible with at least some enzymatic reactions as we have shown thatoligonucleotides tethered to glass through a hexaethylene glycol linkercan be converted to a 5'-phosphomonoester by treatment withpolynucleotide kinase and ATP.

Desirable properties of the Linker

For the applications envisaged, it is desirable that the linker moleculehas the following properties:

It should be possible to link it to a solid support to allow forsynthetic cycles to produce the analyte and corresponding tags toproceed without the need for cumbersome purification of intermediates.Following synthesis cycles, the linker should be removable from thesolid support under conditions which leave the analyte and tags intact.The functional group for tag synthesis should be such that it allows forthe ready synthesis of tags which are distinguishable from each other bymass spectrometry.

The linker should have protected functional groups that allow for theextension of the analyte and the tags separately, under conditions inwhich the chemistry for one does not interfere with that of the other.

The linker should preferably carry a charged group so that massspectrometry can be carried out in the absence of a matrix. Further tothis aim, it is desirable that the tags should comprise compounds whichare volatile enough to evaporate in the mass spectrometer, withoutrecourse to complex techniques such as the electrospray. The tags shouldeither produce stable ions or ions which fragment to characteristicpatterns that can be used to identify the corresponding analyte.

The link between the tag and analyte should preferably bephotocleavable, so that tags can be directly cleaved in the massspectrometer by laser irradiation, and further cleavage to remove themcompletely to allow biochemical steps such as ligation, can be carriedout conveniently by exposure to a lamp.

The linked products should preferably be soluble in aqueous solvents, sothat they can be used in biochemical reactions.

The examples described herein show linkers with these desiredproperties.

Photocleavable Group

The photocleavable group has been based on the known photolabileo-nitrobenzyl group. This group has been used as a protecting group forboth the phosphate group and 2 hydroxy group in oligo nucleotidesynthesis see the review by Pillai Synthesis 1 (1980)!. In itself theo-nitrobenzyl group lacks further functionalisation for subsequentattachment of a linker between tags and analyte. Available fromcommercial sources is the compound 5-hydroxy-2-nitrobenzyl alcohol. Itis known that OMe groups can be added in the 5,4 position withoutsignificant reduction in photolabile properties (see Pillai review).Thus, the 5-hydroxy-2-nitrobenzyl alcohol was used as a starting pointwith the aim of extending DNA synthesis from the benzyl alcohol and thelinker chain to the tags from an ether coupling at the 5-hydroxy group.

The requirement is for a functional group to be present to permit thecombinatorial synthesis of analytes and tags. A linker arm is thereforerequired from the photocleavable group to the required functional groupfor tag synthesis. It is also a preferment that the combinatorialsynthesis be carried out on a solid support. Thus, the linker arm mustbe bivalent in functional groups and have orthogonal protecting groupsto permit selective synthetic transformation. Preferred tag reagentscontain glycol linkages/ether linkages. For synthesis oligonucleotidesare normally linked to a long chain amino CPG support via the 3' hydroxyand a succinic ester link. Thus the functional groups required weredeemed to be alcohols.

The following intermediate compound has been synthesised. ##STR2##

This comprises an aromatic linker carrying:

a methoxytrityl group (--CH₂ ODMT) for analyte synthesis;

an o-nitro-group for photocleavage;

an O-t-butyl diphenyl silyl group (OTBDPS) for tag synthesis;

a tertiary amine group for conversion to a positively charged group foranalysis by mass spectrometry;

and an N-hydroxysuccinimidyl group for attachment to a support.

When the analyte is a peptide only minor modifications to conditionsneed be considered. The 2-nitrobenzyl group is stable under most of theconditions of peptide synthesis and it and related analogues havealready been used as photo labile groups in peptide synthesis (seePillai review and the references contained therein). There are alreadyseveral resins suited to peptide synthesis with different modes ofcleavage. The orthogonal protecting groups for analyte and tag synthesiswould be based on t-butoxycarbonyl and 2-methoxyethoxymethyl. Thet-butoxycarbonyl group would be used to protect the amino group in theamino acids with cleavage being effected by a trifluoroacetic acidtreatment. The 2-methoxyethoxymethyl would be used to protect thetagging groups and the tags based on mass diffentiated on 1, n alkyldiolderivatives as before. The cleavage of t-butoxycarbonyl groups has beenshown to be compatible with the 2-methoxyethoxymethyl protecting groups.The 2-methoxyethoxymethyl protecting groups can be selectively cleavedwith zinc bromide in dichloromethane. While the above illustrates theprocedure those skilled in the art will recognise that this set oforthogonal protecting groups is by no means limiting but serves as arepresentative example.

Detection and analysis of reporters

Photocleavage is the favoured method of releasing tags from analytes; itis fast, can be carried out in the dry state, and scanning lasers can beused to image at a very small scale, small enough to image featureswithin cells (de Vries et al., 1992), so that the proposed method couldbe used to detect the positions of specific analytes that had been usedto "stain" the surface or the insides of cells, or different cells in atissue slice, such as may be required to image interactions betweenligands, e.g. candidate drugs, and their receptors.

Photosensitive protecting groups are available for a very wide range ofchemical residues reviewed in Pillai, 1980!. The photolabile o-nitrobenzyl group which can be used as a protecting group for a wide range ofcompounds forms an ideal starting point for a linker for many analytesthat could be envisaged, peptides and oligonucleotides among them.Taking the example of oligonucleotides, it provides a photosensitivelink that can be broken quantitatively to give a hydroxyl group. Thiswill permit the deprotected oligonucleotide to take place in theligation extension as described in the sequencing method below.Furthermore, the group is known to be stable during oligonucleotidesynthesis. It would be necessary to modify the benzyl ring to provide agroup that can be used to initiate the synthesis of the tags; reporterssuch as the oligoethyleneglycol series described above do not interferewith the photochemical cleavage reaction of the o-nitrobenzoyl group(Pillai op. cit.). Other groups can be added to the aromatic ring whichenhance the cleavage; such groups could be exploited to add a chargedgroup(s) to simplify analysis in the mass spectrometer. Modern massspectrometers are capable of measuring a few hundred molecules with aresolution better than one Dalton in a hundred, up to a total mass of200 kD. A preferred photolabile linker may be represented thus; in whichthe positively charged group R may be directly attached to the aromaticring or may be present in one of the linker arms: ##STR3##

Instrumentation

The proposed molecular tags would be analysed by one of several forms ofmass spectrometry. For many purposes, although it will be desirable tocleave the tags from the analytes, it will not be necessary to fragmentthe tags, and indeed it may be undesirable as it could lead toambiguities. Recent developments in mass spectrometry allow themeasurement of very large molecules without too much fragmentation; andas it is possible to design the linker so that it is readily cleaved,under conditions where the rest of the tag is stable, fragmentation ofthe tag during measurement should be avoidable. The analyte group will,in most cases, be less volatile than the tag, and in many applicationswill be bound to a solid substrate, and thus prevented from interferingwith mass spectrometry.

The linker illustrated above is very labile to photon irradiation underconditions which will cause no cleavage of the great majority ofcovalent chemical bonds. A suitable instrument has been described deVries et al., 1992!. This uses a laser that can be focussed down to aspot smaller than 1 μm. Images of up to 250mm are scanned by moving astage that can be positioned to 0.1 μm.

This instrument also allows for ionisation of the species to be measuredby shining an ionising laser across the surface of the stage so that itinteracts with the species lifted by the desorption laser. This could beuseful for the present method if it were not possible to include acharged residue in the tags, or if fragmentation is desirable forreading the tags.

In another aspect the invention provides a method of sequencing a targetnucleic acid, which method comprises the steps of:

a) providing an oligonucleotide immobilised on a support,

b) hybridising the target nucleic acid with the immobilisedoligonucleotide,

c) incubating the hybrid from b) with the library as defined in whichthe reagents are mixed together in solution, so that an oligonucleotidechain of a first reagent of the library becomes hybridised to the targetnucleic acid adjacent the immobilised oligonucleotide,

d) ligating the adjacent oligonucleotides, thus forming a ligated firstreagent,

e) removing other non-ligated reagents, and

f) recovering and analysing the tag moiety of the ligated first reagentas an indication of the sequence of a first part of the target nucleicacid.

Example applications

We illustrate potential applications by referring to ways in which codedoligonucleotides could be used in nucleic acid analysis.

1. Nucleic acid sequence determination by progressive ligation. (FIG. 4)

The sequence to be determined is first hybridised in step b) to anoligonucleotide attached to a solid support. If the DNA to be sequencedhas been cloned in a single strand vector such as bacteriophage M 13 ,the "primer" oligonucleotide on the solid support can be chosen to bepart of the vector sequence. In step c), the solid support carrying thehybrids from step b) is incubated with a solution of the codedoligonucleotide reagents, e.g. with the aforesaid library, comprisingall sequences of a given length, say 4096 hexanucleotides (4^(n) n-mers,in general). In step d), ligase is introduced so that the hexanucleotidecomplementary to the first six bases in the target DNA is joined to theimmobilised primer oligonucleotide. By this step a first codedoligonucleotide reagent from the library is joined, by ligation of itsoligonucleotide chain to the immobilised primer oligonucleotide, and isherein referred to as a ligated first reagent.

In step e), non-ligated reagents are removed, e.g. by washing. In stepf), the linker of the ligated first reagent is broken to detach the tagchain, which is recovered and analysed as a indication of the sequenceof a first part of the target DNA.

Preferably, removal of the linker also exposes a hydroxyl or phosphategroup at the end of the first oligonucleotide chain, making it availablefor ligation with the oligonucleotide chain of a second reagent. Severalmethods for breaking the linker, including photochemical and enzymaticand chemical hydrolysis, can be used to generate the 3'-hydroxyl or5'-phosphate group needed for further ligation. Steps c), d), e) and f)are then repeated. These steps involve hybridisation of a second reagentfrom the library, ligation recovery and analysis of the tag chain of theligated second reagent, and generation of another 3'-hydroxyl or5'-phosphate group needed for further ligation. The process can berepeated until the whole DNA sequence has been read or until yields inthe reaction become too low to be useful.

Four stages of this sequence are shown diagrammatically in FIG. 4. Thefirst diagram corresponds to the situation at the end of step e) firsttime round. The second diagram corresponds to the situation at the endof step f). The third diagram corresponds to the position at the end ofstep c) second time round. The fourth diagram corresponds to thesituation at the end of step d) second time round.

The cyclic nature of the technique is indicated.

2. Nucleic acid sequencing of multiple templates by sequential ligation.

In an extension of the first example, it is envisaged that manysequences could be analysed simultaneously. For example, individualclones of the DNA to be sequenced could be immobilised:

a) Use can be made of an array of pins with the same vectoroligonucleotide immobilised on the end of each. An individual clone ofthe target DNA is hybridised to the oligonucleotide immobilised on eachindividual pin. The array of pins carrying these hybrids is thenincubated with the library of coded oligonucleotide reagents in asolution which also contains the ingredients for ligation. As a resultof this step, each pin carries a different ligated reagent. Finally, thetag chain of each ligated reagent is recovered and analysed as before.If the pins of the array are suitably spaced, they may be dipped intothe wells of microtitre plates, the first plate containing the templatesto be sequenced, the second the library of reagents and ligationsolution, and the third plate containing a reagent for cleaving the tagchains from the pins.

b) Alternatively, a surface may be coated with the primeroligonucleotide, preferably covalently attached through its 5 end oralternatively at some other point. Individual clones of the DNA to besequenced are spotted at spaced locations on the coated support, so thateach individual clone of the target DNA is hybridised to theoligonucleotide immobilised at an individual spaced location on thesupport. The support is then incubated with a solution containing thelibrary of reagents and the ingredients for ligation. Non-ligatedreagents are removed. Then the linker of the ligated reagent at eachspaced location is cleaved and the tag recovered and analysed.

Cleavage is preferably effected by a method such as laser desorptionwhich can address small areas on the surface. An advantage of thisapproach is that very large numbers of DNA sequences can be analysedtogether.

3. Extension of methods for sequence determination by hybridisation tooligonucleotides

a) Format I.

Methods for spotting DNAs at high density on membranes are wellestablished Hoheisel et al., 1992; Ross et al., 1992!. Forfingerprinting and for sequence determination, oligonucleotides must beapplied either singly or in small sets so that the hybridisationpatterns are not too complex to interpret; as a consequence, only asmall proportion of templates give signal at each round of analysis. Ifthe signal from each hybridisation contained coded information whichallowed its sequence to be determined, more complex mixtures could beused and much more information collected at each round of hybridisation.The complexity of the mixture would depend on the length of the DNAtemplates and on the ability of the analytical method to resolvesequences in mixed oligonucleotides.

Nucleic acid probes encoded with these mass spectrometry tags orreporter groups will be very valuable where the use of multiple probesis advantageous eg. DNA fingerprinting or mutation analysis. The massspectrometry tags offer the advantage of multiplexing.

A number of different probes each labelled with its own unique andappropriate mass spectrometry tag can be used together in typicalnucleic acid hybridisation assays. The sequence of each individual probewhich hybridises can be uniquely determined in the presence of othersbecause of the separation and resolution of the tags in the massspectrum.

In this aspect, the invention provides a method of sequencing a targetnucleic acid, which method comprises the steps of:

i) providing the target nucleic acid immobilised on a support.Preferably individual clones of the target nucleic acid are immobilisedat spaced locations on the support.

ii) incubating the immobilised target nucleic acid from I) with aplurality of the coded oligonucleotide reagents described above, so thatthe oligonucleotide chains of different reagents become hybridised tothe target nucleic acid on the support,

iii) removing non-hybridised reagents, and

iv) recovering and analysing the tag moiety of each reagent as anindication of the sequence of a part of the target nucleic acid.

Preferably thereafter use is made of the library of reagents, with thehybridisation, ligation, cleavage and analysis steps being repeatedcyclically to provide additional information about the sequence of thetarget nucleic acid.

b) Format II

It is possible to determine nucleic acid sequences from the pattern ofduplexes formed when they are hybridised to an array ofoligonucleotides. The length of sequence that can be determined isapproximately the square root of the size of the array: if an array ofall 65,536 octanucleotides is used, the sequences to be determinedshould be around 200 bp Southern et al., 1992!. The limit in size isimposed by the constraint that no run of eight bases should occur morethan once in the sequence to be determined. The array and its use insequence determination are described in International patent applicationWO 89/10977; and a method of providing an array of oligonucleotidesimmobilised e.g. by their 5'-ends or their 3'-ends on a surface isdescribed in International application WO 90/03382.

By the method of the present invention, the sequence length that can bedetermined can be greatly extended. In this aspect of the invention, themethod comprises the steps of:

a) Providing an array of oligonucleotides immobilised at spacedlocations on a support, the oligonucleotide at one location beingdifferent from oligonucleotides at other locations. Preferably thesequence is known of the oligonucleotide immobilised by a covalent bondat each spaced location on the support,

b) incubating the target nucleic acid with the array of immobilisedoligonucleotides, so as to form hybrids at one or more spaced locationson the support,

c) incubating the hybrids from b) with the library of codedoligonucleotide reagents, so that an oligonucleotide chain of a reagentof the library becomes hybridised to the target nucleic acid adjacenteach immobilised oligonucleotide,

d) ligating adjacent oligonucleotides thus forming ligated reagents atthe one or more spaced locations on the support,

e) removing other non-ligated reagents, and

f) recovering and analysing the tag moiety of each ligated reagent as anindication of the sequence of a part of the target nucleic acid.

Preferably cleavage of the tag chain at each spaced location is effectedphotochemically by means of a laser. Preferably analysis of the tagchains is by mass spectrometry. Preferably the hybridisation, ligation,cleavage and analysis steps are repeated cyclically, as described above,so as to obtain additional information about the sequence of the targetnucleic acid.

A preferred sequence of operations is shown in the four diagramsconstituting FIG. 5. The first diagram shows the position at the startof step b). The second diagram shows the position at the end of stepb)--a portion of the target nucleic acid has become hybridised to atethered oligonucleotide forming part of the array. The third diagramshows the position at the end of step c) , and the fourth diagram showsthe position at the end of step d); a reagent from the library hasbecome hybridised to the target nucleic acid and ligated to theimmobilised oligonucleotide.

The results of this extension of the known method are drama tic. Asingle extension by a length equal to the length of the oligonucleotidesin the array squares the overall length that can be read, provided thatthe method used to read the tags can resolve mixtures. In this case thelength that can be read from an array of octanucleotides extended byeight bases is around 60,000 bases.

Comparison of hybridisation analysis with tagged oligonucleotides with:

a) Gel-based methods.

The most advanced instrument for automated sequence analysis is capableof reading around 40000 bases per day. This does not include the timefor the biological and biochemical processes needed to provide thereactions that are loaded on the gel. If we assume that templates can beapplied to a surface at a density of one per square millimeter Hoheiselet al., 1992; Ross et al., 1992!, 10000 could be applied to an area of100×100 mm. After hybridisation, there would be several fmol of taggedoligonucleotide in each cell so a single 2 nsec pulse of the laser mayrelease enough tag to read, but even if we assume that 100 pulses areneeded, then the total time for a cell to be read is a few msec, so thatall 10000 cells could be read in a few minutes. If the oligonucleotideswere hexamers, the raw data acquired would be 60000 bases. For sequencedetermination, this would not be as informative as the equivalent rawdata from a gel, because much longer continuous lengths are read fromgels. This advantage for gels would, of course, be lost if the sequenceread from the array could be extended by further rounds of analysis. Butthe fundamental advantage of array-based approaches is the parallelismwhich enables thousands of templates to be analysed together; the numberthat can be analysed on a gel is limited by the width of the gel to lessthan fifty.

b) Present array-based methods.

The major drawbacks of existing array-based methods are:

a) The sequence that can be read from an array of size N is only ≈√N, sothat most cells of the array are empty. By adding taggedoligonucleotides, the occupancy of the array could be near complete, sothat information would be obtained from most cells. The reason for thisis that additional information from the tags helps remove ambiguitiesdue to multiple occurrences of short strings in the target sequence(Table 3).

b) The length of sequence that is read from each interaction with anoligonucleotide by hybridisation is necessarily limited to the length ofthe oligonucleotide. This causes problems in reading through repeatingsequences, such as runs of a single base. Extending the read by ligationwill permit reads as long as can be traversed by repeated ligations.

c) Of present detection methods, radioactivity has high sensitivity butpoor resolution, fluorescence has low sensitivity and high resolution;both are relatively slow. The proposal to use mass spectrometry couldimprove resolution, speed and sensitivity, as well as adding thepotential to read the sequences of tags.

                  TABLE 3                                                         ______________________________________                                        In general, the sequence that can be                                          determined from templates distributed on a spatially                          segmented array is ≈√4.sup.L = 2.sup.L, where L is the sum     of                                                                            the continuous lengths read by oligonucleotides. This                         would include the length of the oligonucleotide on the                        solid support in example 3b but not in example 2.                                     L   2.sup.L                                                           ______________________________________                                                12  4096                                                                      14  16384                                                                     16  65536                                                                     18  2262144                                                           ______________________________________                                    

Analytes with potential pharmacological activity.

Many drugs are tissue-specific. Their action often depends oninteraction with a cell-surface receptor. There are families of drugsbased on core structures; for example, there are several comprisingshort peptides. It is useful to be able to trace candidate drugs to seewhich cells or tissues they may target. It would be useful to be able totrace many different candidates simultaneously. Using libraries ofanalytes tagged with coded mass-tags, it would be possible to traceinteractions by examining cells or tissues in the mass spectrometer. Iftags were attached by photolabile protecting groups, it would bepossible to image whole animal or tissue sections using scanning lasercleavage, coupled with mass spectrometry.

The following Examples further illustrate the invention.

Examples 1 to 6 show steps, according to the following Reaction Scheme1, in the synthesis of a compound (8) comprising an aromatic linkercarrying: a methyloxytrityl group (--CH₂ ODMT) for analyte synthesis; ano-nitro group for photocleavage; an O-t-butyl diphenyl silyl group(OTBDPS) for tag synthesis; a tertiary amino group for conversion to apositively charged group for analysis by mass spectrometry; and anN-hydroxysuccinimidyl group for attachment to a support.

Examples 7 and 8 show subsequent steps according to the followingReaction Scheme 2.

Examples 9 and 10 show steps, according to the following reaction Scheme3, of preparing reporter groups (13) based on propan-1,3-diol.

Examples 11 to 13 show steps, according to the following Reaction Scheme4, involved in attaching a protected propan-1,3-diol residue as areporter group to compound (6).

Examples 14 to 19 describe the preparation, characterisation and use ofvarious reagents according to the invention.

General Detail

5-Hydroxy-2-nitrobenzyl alcohol was purchased from Aldrich, long chainalkylamino controlled pore glass from Sigma. Anhydrous solvents refer toAldrich Sure Seal grade material packed under Nitrogen. Triethylaminewas predistilled from calcium hydride and stored under nitrogen prior touse. Other solvents and reagents are available from a range ofcommercial sources.

¹ H NMRs were obtained on a Jeol 270 MHz machine using the solventindicated and referenced to tetramethylsilane.

Infra Reds were obtained on a Nicolet 5DXC F.T. IR machine either as apotassium bromide disc or chloroform solution as indicated.

Melting points were obtained on a Gallenkamp melting point apparatus andare uncorrected.

Tics were run on Kieselgel 60F₂₅₄ aluminium backed Tic plates using thesolvent system indicated. The plates were visualised by both ultraviolet and/or dipping in a 3% w/v ethanolic solution ofmolybdophosphoric acid and then heating with a hot air gun. Tritylcontaining species show up as a bright orange spot, alcohols as a bluespot.

Silica gel chromatography was performed using flash grade silica gel,particles size 40→63 μm.

Abbreviations used in the reaction schemes and text.

DMT--4,4¹ -dimethoxytrityl

THF--tetrahydrofuran

TBDPS--tert-butyldiphenylsilane

DMAD--4-dimethylaminopyridine

DCCI--dicyclohexyidicarbodiimide

CH₂ Cl₂ --dichloromethane

CPG--controlled pore glass

Mel--iodomethane

Tresyl--2,2,2-trifluoroethlsulphonyl

EXAMPLE 1 Synthesis of S-hydroxy-O-(4,4¹ -dimethoxytrityl)-2-nitrobenzylalcohol (Compound 2, Scheme 1)

To 5-hydroxy-2-nitrobenzyl alcohol (5.11 g, 30.2 mmol) dissolved inanhydrous pyridine (40 ml) was added 4,4¹ -dimethoxytrityl chloride(10.25 g, 30.2 mmol) and the flask stoppered. The reaction mixture wasthen left to stir at room temperture for a total of 72 hours. T.l.c.analysis (ether/pet. ether 40°-60° C., 65%135%) revealed the presence ofa new trityl positive containing material with an R_(F) of 0.27 anddisappearance of the starting alcohol. The pyridine was then removed byrotary evaporation, with the last traces being removed by co-evaporationwith toluene (x2). The resultant gum was dissolved up in ethyl acetateand the solution washed with water (x1) and brine (x1). The ethylacetate solution was then dried over anhydrous magnesium sulphate andevaporated to a reddish brown gum. The gum was dissolved in CH₂ Cl₂ (20ml) and then applied to a silica gel column (14 cm×6.5 cm) which waseluated with ether/ pet. ether 40°-60° C., 65 %/35%. The productfractions were combined and the solvent removed by rotary evaporation togive an off white solid (13.49 g, 95%, mpt. 80°-82° C. withdecomposition). An analytical sample was prepared by recrystallisationfrom chloroform/ pet. ether 40°-60° C., mpt. 134°-7° C. withdecomposition.

¹ H NMR (270 MHz, CDCl₃ δ): 3.79 (s, 6H, DMT-OCH₃), 4.63 (s, 2H, CH₂-ODMT), 6.77-6.85 (m, 5H, aryl), 7.22-7.49 (m, 9H, aryl), 7.63 (s, 1Haryl), 8.06 (d, 1H, J=9.06 Hz, aryl).

IR (KBr disc), 1610, 1509, 1447, 1334, 1248, 1090, 1060, 1033, 828 cm⁻¹.

EXAMPLE 2 Synthesis of O-(4,4¹ -dimethoxytrityl)-5-1-(3-bromo-1-oxypropyl)!-2-nitrobenzylalcohol (Compound 3, Scheme 1)

To compound 2 (10.18 g, 21.6 mmol) dissolved in acetone (150 ml) wasadded 1,3-dibromopropane (11 mls, 108 mmol) and potassium carbonate(4.47 g, 32.3 mmol). The reaction mixture was then heated at 80° C. fora total of three hours and then stirred at room temperature for afurther 16 hours. T.l.c. analysis (ether/pet. ether 40°→60° C., 60%/40%)showed complete disappearance of the starting material and the formationof two new trityl containing species; R_(F) 0.48 major, R_(F) 0.23minor. The acetone was then removed by rotary evaporation and theresultant residue partitioned between water and dichloromethane. Thedichloromethane solution was separated and washed with brine. Thedicholormethane solution was then dried over anhydrous magnesiumsulphate and evaporated down to a gum. The gum was dissolved indichloromethane 20 ml and then applied to a silica gel column (6.5 cm×14cm) which was eluated with ether/pet. ether 40°-60° C., 60%140%. Thepure product fractions were combined and the solvent removed by rotaryevaporation to give compound 3 as a white solid (8.18 g, 64%, mpt.132°→4° C., R_(F) 0.48 ether/pet ether 40°-60° C., 60%/40%. A smallsample was recrystallised from ethyl acetate/pet. ether for analyticalpurposes, mpt. 132°-4° C.

¹ H NMR: (270 MHz CDCl₃, δ): 2.40 (m, 2H, --CH₂ --CH₂ --CH₂ --), 3.64(t, 2H, J=6.32 Hz, CH₂ Br), 3.79 (s, 6H, OCH3), 4.27 (t, 2H, J=6.04Hz,-OCH₂ CH₂), 4.66 (s,2H, Ar CH₂ ODMT), 6.84 (d, 4H, J=8.79 Hz, DMTaryl), 7.20-7.50 (m, 10H,9 DMT aryl, 1 aryl) 7.68 (s, 1H, aryl), 8.1(d,1H, J=9.06 Hz, aryl).

IR (KBr disc) 1608, 1577, 1511, 1289, 1253, 1230, 1174, 1065, 1030 cm⁻¹.

EXAMPLE 3 Synthesis of N-O-(tert-butyldiphenylsilyl)-2-oxyethyl)!-N-(2-hydroxyethyl) amine(Compound S, Scheme 1)

To sodium hydride (0.76 g of a 60% dispersion in oil, 19 mmol) under N₂was added anhydrous THF (15 ml)) followed by a slurry of diethanolamine(2g, 19 mmol) in THF (30 ml) at such a rate as the evolution of hydrogenpermitted. The reaction mixture was then stirred at room temperature for30 minutes under N₂ during which time a grey precipitate formed. Thegenerated alkoxide was quenched by the addition oftert-butylchlorodiphenylsilane (4.95 ml, 19 mmol) followed by stirringthe reaction at room temperature for two hours under N₂. T.l.c. analysis(ethyl acetate) showed the generation of two new UV positive spotsrelative to starting material, major R_(F) 0.05 minor R_(F) 0.60. TheTHF was removed by rotary evaporation and the residue dissolved in a0.1M sodium bicarbonate solution. The product was then extracted withethyl acetate (x2). The ethyl acetate extracts were then combined andwashed with brine (x1). The ethyl acetate solution was then dried overanhydrous magnesium sulphate and evaporated down to an oil. This oil wasapplied to a silica gel column which was elulated with achloroform/methanol, 90%/10% Fractions with an R_(F) of 0.33 werecombined and rotary evaporated to give compound 5 as a white crystallinesolid (3.93 g, 60%, mpt. 73°→75° C.). A small sample was recrystallisedfrom ethyl acetate/ pet. ether 40°-60° C. for analytical purposes, mpt.76°→77° C.

¹ H NMR (270MHz, CDCl₃, δ): 1.06 (s, 9H, 'Bu), 2.13 (brs, 1H, OH,D₂ Oexchangable), 2.78 (m, 4H, CH₂ NHCH--₂), 3.63 (t, 2H, J=5.22 Hz, --CH₂OSi--), 3.78 (t,2H, J=5.22 Hz,CH₂ OH), 7.40 (m, 6H, aryl), 7.66 (m, 4H,aryl).

IR (KBr disc) 3100, 1430, 1114, 1080, 969, 749, 738, 707 cm⁻¹.

EXAMPLE 4 Synthesis of N- O-(tert-butyldiphenylsilyl)-2-oxyethyl! -N-O-(3(O-(4,4¹-dimethoxytrityl)-1-oxymethyl)-4nitrophenyl)-3-oxypropyl!-N-(2-hydroxyethyl)amine(Compound 6, Scheme 1)

To compound 3 (7.46 g, 12.6 mmol) dissolved in 1-methyl-2-pyrrolidinone(65 ml) was added compound 5 (8.63 g, 25.2 mmol). The reaction mixturewas then heated at 80° C. for a total of 5 hours before being left tocool and stir at room temperature for a further 16 hours. T.l.c.analysis (ethyl acetate) showed the formation of a new trityl containingspecies, R_(F) 0.51 and residual amounts of the two starting materials.The reaction mixed was poured into a mixture of water (600 ml) and brine(100 ml) and the product extracted with ethyl acetate (3×200 ml). Theethyl acetate extracts were then combined and dried over anhydrousmagnesium sulphate. The ethyl acetate was then removed by rotaryevaporation to give a brown gum from which a crystalline product slowlyformed. The minimum amount of ethyl acetate was added to dissolve up theresidual gum such that the crystalline product could be filtered, thehydrogen bromide salt of compound 5. The ethyl acetate solution was thenapplied to a silica gel column (13 cm×6.5 cm) which was eluted withethyl acetate. Insufficient separation of residual compound 3 and thedesired product was obtained from this column so the product fractionswere combined and evaporated to a gum. This gum was dissolved up in theminimum of ethyl acetate necessary and applied to another silica gelcolumn (14 cm×6.5 cm) eluting using a gradient eluation, first ethylacetate/pet. ether 40°→60° C., 50%150% followed by ethyl acetate. Theproduct fractions were combined and the solvent removed by rotaryevaporation to give compound 6 as a gum. The last traces of solvent wereremoved by placing the gum under high vacuum for one hour. The yield ofproduct was 7.64 g, 71%.

¹ H NMR (270 MHz, CDCl₃, δ): 1.04 (s, 9H, 'Bu), 1.97 (m, 2H, --CH₂ CH₂CH₂ --), 2.7 (m, 6H, NCH₂), 3.56 (m, 2H, CH₂ OH), 3.75 (m, 2H, CH₂ OSi),3.78 (s, 6H, DMT-OCH₃), 4.12 (m,2H, ArOCH₂ CH₂), 4.64 (s, 2H, ArCH₂ODMT), 6.74-6.85 (m,5H,aryl) 7.2-7.65 (m, 20H, aryl), 8.05 (d,1H, aryl).

IR (KBr disc), 1608, 1579, 1509, 1287, 1251, 1232, 1112, 1092, 1064,1035, 826, 754, 703, 613 cm⁻¹.

EXAMPLE 5 Synthesis of N- O-(tert-butyldiphenylsilyl)-2-oxyethyl!-N-O-(3-(O-(4,4¹-dimethoxytrityl)-1-oxymethyl)-4-nitrophenyl)-3oxypropyl!-N-O-(3-carboxylatopropionyl))-2-oxyethyl!amine (Compound 7, Scheme 1)

To compound 6 (5.64 g, 6.59 mmol) dissolved in anhydrous dichloromethane(40 ml) and anhydrous pyridine (50 ml) was added succinic anhydride(2.06 g 20.6 mmol) and dimethylaminopyridine (210 mg, 1.72 mmol) and theflask stoppered. The reaction was then stirred at room temperature for atotal of 72 hours. T.l.c. analysis (methanol/ethyl acetate, 10%/90%)showed the formation of a new trityl containing species, R_(F) 0.45 andthe disappearance of the starting material. The solvent was removed byrotary evaporation with the last traces of pyridine being removed byco-evaporation with toluene (x2). The resultant gum was then partitionedbetween chloroform and water. The organic phase was separated and theaqueous phase further extracted with chloroform (x1). The organic phaseswere then combined and washed with brine (x1). The chloroform solutionwas then dried with anhydrous magnesium sulphate and evaporated to agum. The last traces of solvent were then removed by placing the gumunder high vacuum for one hour to give compound 7, 6.75 g. This productwas used in the next step without further purification.

¹ H NMR (270 MHz, CDCl₃, δ): 1.0 (s, 9H, 'Bu), 1.9 (m, 2H, CH₂ CH₂ CH₂),2.5 (m, 4H, COCH₂ CH₂ COOH), 2.7 (m, 6H, N-CH₂), 3.7 (m, 2H, CH₂ OSi),3.75 (s, 6H, DMT OCH₃), 4.1 (m, 4H, CH₂ OCO and Ar-OCH₂ CH₂, 5.6 (s, 2H,ArCH₂ ODMT), 6.7 (d, 1H, aryl), 6.8 (d,4H, aryl) 7.2→7.7 (m, 20H, aryl),8.02 (d, 1H, aryl).

IR (CHCl₃ solution) 1736, 1608, 1579, 1509, 1288, 1251, 1232, 1175,115.8, 1112, 1093, 1065, 1035, 755, 703 cm⁻¹.

EXAMPLE 6 Synthesis of N- O-tert-butyldiphenylsilyl)-2-oxyethyl!-N-O-(3-(O-(4,4-dimethoxytrityl)-1-oxymethyl)-4-nitrophenyl)-3-oxypropyl!-N-(O-(succinyl (3-carboxylatopropionyl)))-2-oxyethyl!amine (Compound 8,Scheme 1)

To compound 7 (2.99 g, 3.13 mmol) dissolved in anhydrous dichloromethane(30 ml) was added dicyclohexylcarbodiimide (0.710 g, 3.45 mmol) andN-hydroxy succinimide (0.396 g, 3.44 mmol) and the flask stoppered. Thereaction mixture was then allowed to stir at room temperature for 18hours during which time a white precipitate formed. The whiteprecipitate was filtered off and the dichloromethane solution washedwith water (x1) and brine (x1). The dichloromethane solution was thendried over anhydrous magnesium sulphate and the solvent rotaryevaporated off to give a foam, 3.26 g (99%). T.l.c. analysis (ethylacetate) showed only one trityl containing species, R_(F) 0.74 and nosignificant containment. Attempts to provide an analytical sample bypassing a small amount of material down a silica gel column resulted inthe decomposition of the active ester back to the acid (Compound 7). Thematerial was therefore used in all further equipments without furtherpurification.

¹ H NMR (270 MHz, CDCl₃, δ): 1.04 (s, 9H, 'Bu), 1.97 (m, 2H, CH₂ CH₂CH₂), 2.50→2.75 (m, 6H, succinyl CH₂ +--OCCH₂), 2.76-2.86 (m, 6H, NCH₂),3.08 (m, 2H, CH₂ CO₂ succinyl), 3.77 (s, 6H, DMTOCH₃), 3.86 (m, 2H, CH₂OSi), 4.1→4.2 (m, 4H, AROCH₂ +CH₂ O₂ C), 4.63 (s, 2H, ArCH₂ ODMT,6.7→6.9 (m, 5H, aryl), 7.01→7.7 (m, 20H, aryl) 8.05 (d, 1H, aryl).

IR (KBr disc), 1742, 1713, 1509, 1288, 1251, 1211, 1175, 1090, 1067cm⁻¹. ##STR4##

EXAMPLE 7 Derivatised long chain alkylamino controlled pore glass(Compound 9, Scheme 2)

Long chain alkylamino controlled pore glass (Sigma Chemical Co, 3.5 g)was pre-treated with trichloroacetic acid (1.5 g) dissolved indichloromethane (50 ml) for 21/2 hours, washed with aliquots ofdichloromethane (100 ml total) and anhydrous ether (100 ml total) anddried in vacuo. To the CPG support was then added anhydrous pyridine (35ml), dimethylaminopyridine (42 mg, 344 μmol) triethylamine (280 μl, 201mmol) and compound (8) (see scheme 1) (736 mg, 700 μmol). The mixturewas then gently agitated for a total of 18 hours after which time thebeads were given multiple washes of pyridine (7×10 ml), methanol (5×15ml) and chloroform (5×15 ml) and then dried in vacuo.

EXAMPLE 8 Methylation of the tertiary amino groups attached to the CPGsupport (Compound 10, Scheme 2)

To the derivatised long chain alkylamino controlled pore glass (Compound9, Scheme 2) (1.01 g) was added anhydrous THF (10 ml) and iodomethane(0.5 ml, 8 mmol). The mixture was then gently agitated for a total of 18hours after which time the beads were given multiple washes of anhydrousTHF (5×10 ml) and then dried in vacuo. ##STR5##

EXAMPLE 9 Synthesis or mono protected 1,3-propanediol derivatives(Compounds 12a and 12b, Scheme 3)--general procedure

To sodium hydride (1.05 g of a 60% dispersion in oil, 26.3 mmol) underN₂ was added anhydrous THF (10 ml) followed by dropwise addition of the1,3-propanediol derivative (26.3 mmol) dissolved in anhydrous THF (20ml). Stirring for an additional 30 minutes under N₂ ensured alkoxideformation as noted by the formation of a grey precipitate. The generatedalkoxide was quenched by the dropwise addition oftert-butylchlorodiphenylisilane (7.24 g, 26.3 mmol) dissolved inanhydrous THF (20 ml) followed by stirring of the reaction under N₂ fora further 40 minutes. The THF was then removed by rotary evaporation andthe residue partitioned between dichloromethane and 0.1M sodiumbicarbonate solution. The dichloromethane solution was separated off andwashed with brine (x1). The dichloromethane solution was then dried overmagnesium sulphate and evaporated down to an oil. This oil was appliedto a silica gel column (16 cm×5 cm) which was eluted with an ether/pet.ether 40°→60° C., 30%/70% mixture. The product fractions were combinedand rotary evaporated down to provide the desired 1,3-propanediolderivative.

For individual details of the compounds see below.

12a 1-O-tert-butyldiphenyslyl-1,3-propanediol, white crystalline solid,R_(F) 0.21 ether/pet. ether 40°→60° C., 30% 70%, 7.61 g, 92%, mpt.40°→42° C.

IR (KBr disc) 3400, 1448, 1112, 822, 734, 702, 689, 506, 488 cm⁻¹.

¹ H NMR (270 MHz, CDCl₃ δ): 1.06 (s, 9H, 'Bu), 1.80 (m,2H, CH₂ CH₂ CH₂),2.45 (t, 1H, OH), 3.84 (m, 4H,OCH₂ CH₂ CH₂ O--), 7.40 (m, 6H, aryl),7.68 (m,4H, aryl).

12b 2-methyl-1-O-tert-butyldiphenylsilyl-1,3-propanediol. Colourlessoil, R_(F) 0.21 ether/pet. ether 40°→60° C., 30%/70%, 6.60 g, 77%.

IR (thin film) 3400, 1472, 1428, 1087, 1040, 823, 740, 702 cm⁻¹.

¹ H NMR (270 MHz, CDCl₃, δ): 0.82 (d, 3H, i =6.87 Hz, CH₃), 1.06 (s,9H,'Bu), 2.0 (m, 1H, CH--CH₃), 2.68 (t, 1H, OH), 3.64 (m, 4H, CH₂ CH (CH₃)CH₂), 7.40 (m,6H, aryl), 7.68 (m, 4H, aryl).

See P G McDougal et al JOC, 51, 3388 (1986) for general procedures forthe monosilylation of symmetric l,n-diols.

EXAMPLE 10 Synthesis of the treslate derivatives (Compounds 13a and 13b,Scheme 3)-general procedures

To the alcohol derivative (4.94 mmol) dissolved in anhydrousdichloromethane (10 ml) and dry triethylamine (0.84 ml 6.03 mmol) underN₂ and cooled to between -15°→30° C. was added the tresylchloride (1 g,5.48 mmol) in anhydrous dichloromethane (5 ml) dropwise over a 20→40minutes. Stirring for an additional 30 minutes under N₂ at -15°→30° C.completed the reaction. The reaction mixture was then transferred to aseparatory funnel and washed with ice cooled 1.0M hydrochloric acid(x1), ice cooled water (x1) and ice cooled brine (x1). Thedichloromethane solution was then dried over magnesium sulphate and thesolvent rotary evaporated off to give the treslate. The treslates werestored at -20° C. under N₂ until required.

For individual details of the compounds see below.

13a 1-O-tert-butyldiphenylsilyl-3-O-tresyl-1,3-propanediol. Whitecrystalline solid, 1.74 g, 77% mpt. 34°→35° C. Three ml of this reactionmixture was removed prior to work up of the reaction for addition toother reactions.

¹ H NMR (270 MHz, CDCl₃, δ): 1.06 (s, 9H, 'Bu), 1.97, (m, 2H, CH₂ CH₂CH₂), 3.77 (t, 2H, J=5.49 Hz, CH₂ --O)--Si), 3.84 (q, 2H, J=8.79 Hz, CF₃--CH₂ --O), 4.54 (t, 2H, J=6.05 Hz, Tresyl O--CH₂), 7.42 (m, 6H, aryl),7.64 (m, 4H, aryl).

IR (KBr disc) 1386, 1329, 1274, 1258, 1185, 1164, 1137, 1094, 941, 763,506 cm⁻¹.

13b 2-methyl-1-O-tert-butyldiphenylsilyl-3-O-tresyl-1,3-propanediol.Colourless oil,2.57 g, 99%.

¹ H NMR (270 MHz, CDCl₃, δ): 0.97 (d, 3H, J=6.87 Hz, CH₃), 1.06 (s, 9H,'Bu), 2.10 (m, 1H, CHCH₃), 3.6 (m, 2H, CH₂ OSi), 3.8 (q, 2H, J=8.79 Hz,CF₃ CH₂), 4.40 (m, 2H, Tresyl-O-CH₂), 7.40 (m, 6H, aryl), 7.64 (m, 4H,aryl).

For general details of Treslates see R K Crossland et al JACS, 93, 4217(1971). ##STR6##

EXAMPLE 11 Synthesis of N- acetoxy-2-oxyethyl!-N- O-(3(O-(4,4¹-dimethoxytrityl)-1-oxymethyl)-4-nitrophenyl)-3-oxypropyl!-N-2-hydroxyethyl!amine(Compound 15, Scheme 4)

To compound 11 (1.72 g, 1.92 mmol) dissolved in anhydrous THF (20 ml)was added tetrabutylammonium fluoride (0.55 ml of a 1M solution in THF,1.92 mmol). The reaction was then stirred for a total of two hours atroom temperature. The reaction mixture was then diluted with water (50ml) and the THF removed by rotary evaporation. The aqueous solution wasthen extracted with chloroform (x1). The organic solution was dried overanhydrous sodium sulphate and evaporated down to a gum. The product waspurified by silica gel chromatography eluting the column with ethylacetate. Product fractions were combined and rotary evaporated down togive compound 12 as a colourless gum which slowly crystallised onstanding; 0.73 g, 58%, mpt. 95°→97° C., R_(F) 0.26 ethyl acetate.

¹ H NMR (270 MHz, CDCl₃, δ): 1.75 (brs, 1H, OH), 2.0→2.1 (m, 5H, O₂ CCH₃+CH₂ CH₂ CH₂), 2.70→2.81 (m, 6H, CH₂ N), 3.58 (m, 2H, CH₂ OSi), 3.79 (s,6H, DMT-OCH₃), 4.17 (m, 4H, CH₂ O), 4.64 (s, 2H, ArCH₂ ODMT), 6.83 (d,4H, DMT-aryl) 7.2→7.5 (m, 1OH, aryl), 7.69 (S, 1H, aryl), 8.10 (d, 1H,aryl).

IR (KBr disc), 3459, 1738, 1608, 1577, 1506, 1444, 1313, 1288, 1250,1230, 1175, 1154, 1070, 1035, 984 cm⁻¹.

EXAMPLE 12 Synthesis or N- O-(tert-butyldiphenylsilyl)-2-oxyethyl!-N-O-(3(O-(4,4'-dimethoxytrityl)-1-oxymethyl)-4-nitrophenyl)-3-oxypropyl!-N-acetoxy-2-oxyethyl!amine (Compound 14, Scheme 4)

To compound 6 (1.73 g, 2.02 mmol) dissolved in anhydrous pyridine (10ml) was added acetic anhydride (0.5 ml, 4.54 mmol) and4-dimethylaminopyridine (55 mg, 0.45 mmol) and the flask stoppered. Thereaction mixture was then stirred at room temperature for a total of 16hours after which time t.l.c. analysis (methanol/ethyl acetate 5%/95%)showed the complete disappearance of the starting material and theformation of a new trityl containing Spot, R_(F) 0.80. The pyridine wasremoved by rotary evaporation with the last traces being removed withco-evaporation with toluene (x2). The resultant gum was partitionedbetween chloroform and water. The chloroform solution was separated offand washed with brine (x1). The chloroform solution was then dried overanhydrous magnesium sulphate and the solvent rotary evaporated off togive a colourless gum, 1.94 g. This material was pure enough to be usedin the next reaction without any further purification.

¹ H NMR (270 MHz, CDCl₃, δ): 1.04 (s, 9H, 'Bu), 1.9 (m, 2H, CH₂ CH₂CH₂), 2.01 (s, 3H, --O₂ CCH₃), 2.74 (m, 6H, CH₂ N), 3.7 (m, 2H, CH₂OSi), 3.8 (s, 6H, DMT-OCH₃), 4.1 (m,4, CH₂ O), 4.63 (s, 2H, ArCH₂ ODMT),6.78 (d, 1H, aryl) 6.83 (d, 4H, DMT aryl), 7.2→7.8 (m 20H aryl), 8.05(d, 2H, aryl)

EXAMPLE 13 Synthesis of N- acetoxy-2-oxyethyl!-N- O-(3(O-(4,4¹-dimethoxytrityl)-1-oxymethyl)4-nitrophenyl)-3-oxypropyl!-N-O-(tert-butyldiphenylsilyl)-3-oxo-6-oxyhexyl!amine (Compound 16, Scheme4)

To compound 12 (66 mg, 0.10 mmol) dissolved in anhydrous acetonitrile (5ml) was added potassium carbonate (55 mgs, 0.4 mmol and compound 13a (1ml of the reaction mixture, approximately 0.30 mmol) and the flask thenstoppered with a calcium chloride drying tube. The reaction mixture wasthen stirred at room temperature for a total of 22 hours after whichtime the potassium carbonate was filtered off and the solvent removed byrotary evaporation. The resultant oil was then applied to a silica gelcolumn (14 cm×1 cm) and the product eluted off with an ether/pet. ether40°→60° C., 75%/25% mixture. The pure product fractions were combinedand evaporated down to a clear gum, 6 mg, 6%, R_(F) 0.47 in ether/pet.ether 40°→60° C., 80%/20%.

¹ H NMR (270 MHz, CDCl₃, δ): 1.05 (s, 9H, 'Bu), 1.8 (m, 2H, CH₂ CHCH₂OSi), 1.9 (m, 5H, O₂ CCH₃ +ArOCH₂ --), 2.76-2.92 (m, 6H, CH₂ N), 3.51(t, 2H, J=6.6 Hz, OCH₂ CH₂ CH₂ OSi), 3.79 (s, 6H, DMT-OCH₃) 3.85 (m, 2H,CH₂ OSi), 4.12→4.23 (m, 4H, ArOCH₂ CH₂ +NCH₂ CH₂ OCOCH₃), 4.64 (s, 2H,ARCH₂ ODMT), 6.83 (m, 5H, 1 aryl+DMT-aryl), 7.23→7.50 (m, 16H, aryl),7.68 (m, 4H, aryl), 8.10 (d, 1H, J=9.06Hz, aryl).

By analogues reaction conditions to the above the following compound hasalso been synthesised utilising the treslate 13b.

N- acetoxy-2-oxyethyl!-N- O-(3(O(4,4¹ -dimethoxytrityl)-1-oxymethyl)-4-nitrophenyl)-3-oxypropyl!-NO-(tert-butyldiphenylsilyl)-5-methyl-3-oxo-6-oxyhexyl!amine. Thecompound is a clear gum, R_(F) 0.53 in ether/pet. ether 40°→60° C.,80%/20%.

¹ H NMR (270 MHz, CDCl₃, δ): 0.88 (d, 3H, CH-CH₃), 1.00 (s, 9H, 'Bu),1.9→2.1 (m, 6H, O₂ CCH₃ +CH--CH₃ +CH₂ CH₂ CH₂), 2.7→3.0 (m, 6H, CH₂ N),3.4→3.7 (m, 4H, CH₂ O--), 3.79 (s, 6H, DMT-OCH₃), 4.0→4.4 (m, 6H, CH₂O--), 4.64 (s, 2H, Ar CH₂ ODMT), 6.83 (m, 5H, aryl), 7.2→7.7 (m, 20H,aryl), 8.01 (d, 1H, aryl). ##STR7##

EXAMPLE 14 Synthesis of oligonucleotides on solid supports

Controlled pore glass carrying linkers 9 and 10 (compounds 9 and 10 inScheme 2) was loaded into the columns used in the automaticoligonucleotide synthesiser (ABI 381A); the amounts used provided for0.2 or 1 μmol scale synthesis. The columns were inserted in theautomatic synthesiser which was then programmed for appropriate cycles.Two different types of nucleotide precursors were used: normalphosphoramidites, with dimethoxytrityl protecting groups on the 5hydroxyls; "reverse synthons" with 5' phosphoramidites anddimethoxytrityl protecting groups on the 3' hydroxyls. A list ofoligonucleotides synthesised on these supports in shown in Table 4 inwhich R9 and R10 derive from compounds 9 and 10 respectively. Yieldswere monitored from the amount of dimethoxytrityl group released at eachcoupling. These yields corresponded to those obtained on the CPGsupports used for conventional oligonucleotide synthesis.

                  TABLE 4                                                         ______________________________________                                        End group(s)                                                                           Sequence  Normal direction                                                                           Reverse direction                             ______________________________________                                        R9       T.sub.5                √                                      R10      T.sub.5                √                                      R10, DMT T.sub.5                √                                      R9, DMT  T.sub.5   √                                                   R10, DMT T.sub.5   √                                                   R10      .sub. A.sub.10                                                                          √                                                   ______________________________________                                    

EXAMPLE 15 Synthesis of tags under conditions which leave the analyteintact

After synthesising 5'R9T₅ on support 9, the solid support was divided,part was treated with 5 mM tetrabutylammonium fluoride in THF for 10min. at room temperature to remove the t-butyldiphenylsilyl protectinggroup. Both samples were treated with 29% ammonia at room temperatureovernight to remove the products from the solid support. Ammonia wasremoved under vacuum, and the solid residue dissolved in water. HPLCshowed the successful removal of the silyl protecting group withretention of the DMT group. This example shows that the two protectinggroups can be removed under conditions which leave the other in place;and further, that removal of the protecting groups leaves theoligonucleotide chain intact.

EXAMPLE 16 Biochemical reactions of tagged analytes

16a. Enzymatic phosphorylation of tagged oligonucleotides. For manypurposes, it will be useful to have oligonucleotides which have aphosphate group at the 5' end. Such a group is necessary if theoligonucleotide is to be used as the donor in a ligation reaction; andit is a useful way of introducing a radioactive group to testbiochemical properties.

The oligonucleotides A₅, A₁₀, and T₅ were made with the tags R9 and R10attached to the 3' ends, with and without the silyl protecting groupremoved (This was achieved by treating the oligonucleotide, still on thesolid support, with a 5 mM solution of tetrabutylammonium fluoride inacetonitrile, at room temperature for 15 min.) These oligonucleotideswere phosphorylated using T4 polynucleotide kinase and gamma-³³ P-ATPusing standard protocols recommended by the supplier. Thin layerchromatography of the products on polyethyleneimine (PEI) impregnatedcellulose developed in 0.5M ammonium bicarbonate showed in each casethat the labelled phosphorus had been transferred almost completely tothe oligonucleotide.

16b. Ezymatic ligation of tagged oligonucleotides.

For some applications of tagged oligonucleotides, it will be useful toligate them to a receptor. We have shown that tagged oligonucleotidescan take place in enzymatic ligation by the following tests:

(1) Oligonucleotides tagged at the 5'-end. In this test, the templatewas 5' ATCAAGTCAGAAAAATATATA (SEQ ID No. 1). This was hybridised to thedonor, 3' TAGTTCAGTC (SEQ ID No. 2), which had been phosphorylated atits 5'-end using radioactive phosphorus. Four ligation reactions werecarried out, each with a modification of the sequence T₅, which couldligate to the 5' phosphorylated end of the donor after hybridising tothe run of 5 A's in the template. The four oligoT's used in thereactions differed in the nature of their 5'-end. One had adimethoxytrityl group attached through the hydroxyl. The second andthird had tags R9 and R10 attached to the 5'-end through aphosphodiester bond. The fourth was a positive control, with a normal5'OH. A negative control lacked any oligoT. Ligation reactions wereperformed using T4 ligase according to the suppliers instructions.Reactions were analysed by TLC on PEI-cellulose, developed in 0.75Mammonium bicarbonate solution. All four reactions showed an additionalspot on the chromatogram, of lower mobility than the donor; as expected,the negative control showed no additional spot. This illustrates howoligonucleotides with different tags can take part in sequence-specificligation reactions.

Cozzarelli et al (1967) have shown that polynucleotides attached tosolid supports can be ligated to an acceptor in the presence of acomplementary template.

EXAMPLE 17 Hybridisation of tagged oligonucleotides to oligonucleotidestethered to a solid support

Example 16b shows that tagged oligonucleotides can take part in ligationreactions, inferring that they can also take part in duplex formation insolution, as ligation depends on this process. The following experimentshows that they can also form duplexes with oligonucleotides tethered toa solid support. T₁₀ was synthesised on the surface of a sheet ofaminated polypropylene according to the manufacturer's instructions. Itis known that this process yields around 10 pmols of oligonucleotide permm². A solution in 3.5M tetramethylammonium chloride of A₁₀ (65 pmol permicrolitre), labelled at the 5'-end with ³³ P, and tagged at the 3 withR10 was laid on the surface of the derivatised polypropylene and leftovernight at 4°. After washing in the hybridisation solvent, it wasfound that around one third of the probe had hybridised to the tetheredoligo-dT. This is close to the theoretical limit of hybridisation,showing that tagged oligonucleotides can take part in hybridisationreactions with high efficiency.

EXAMPLE 18 Photolysis of tags

The potential to remove tags by photolysis would greatly enhance theirusefulness: it would allow for direct analysis by laser desorption inthe mass spectrometer; it would provide a simple method of removing thetags to allow other biochemical or chemical processes.

18a. Bulk photolysis.

The nitrobenzyl group is known to be labile to irradiation at 305 nm.Solutions of R10A₁₀ and R10T₅ in water were irradiated at 2 cm. from atransilluminator for 20 min. under conditions known to cause nodetectable damage to nucleic acids. Analysis by HPLC showed the expectedproducts of photocleavage, with no detectable residue of the originalcompound.

18b. Laser induced photolysis in the mass spectrometer.

Samples of R10T₅ and T₅ R10 were deposited on the metal target of a timeof flight mass spectrometer (Finnigan Lasermat) without added matrix.The spectrum showed a single saturated peak at around mass 243 in thepositive mode that was absent in other samples.

EXAMPLE 19 Identification by mass spectrometry of different tagsattached to different analytes

A sequence of five thymidine residues with a dimethoxytrityl groupattached as a tag to the 3 end was synthesised by conventional solidphase methods, but using "reverse synthons". In the mass spectrometer,this compound gave a large and distinct peak at mass 304, in thepositive ion mode. By contrast a sequence of ten adenosine residuescarrying the tag designated R10 above gave a large and distinct peak atmass 243 in the positive ion mode. In both cases, laser desorption wascarried out in the absence of matrix. In both cases the peaks are absentfrom the oligonucleotides which have no tag. These examples show that itis easily possible to identify an analyte sequence from the presence ofa peak in the mass spectrometric trace that derives from a tagincorporated during the synthesis of the analyte, and thatcharacteristic tags are readily identified by their different mass.

FIGURE LEGENDS

FIG. 1. General scheme for synthesis of molecules with specific tags.

Synthesis starts from a linker (L) with at least one site for theaddition of groups for synthesising the analyte and one for synthesisingthe tag. (The linker may also be attached reversibly to a solid supportduring synthesis, and may have sites for generating groups such ascharged groups which may help in analysis). P_(a) and P_(r) aretemporary protecting groups for the analyte precursors and the reportersrespectively; they will be removable by different treatments. Forexample, P_(a) may be an acid or base labile group such as trityl, F-MOCor t-BOC, and P_(r) a group removable by treatment with fluoride such asa silyl residue. Groups U-Z may also have protecting groups which mustbe stable to the reagents used to remove P_(a) and P_(r). Couplingchemistries will be different for different analyte types; standardmethods are available for oligonucleotide and peptide synthesis.

Three different types of tags are described in FIG. 2. For the firstscheme, each extension of the tag is carried out with a reporter whichis specific for both position and type of residue added to the analyte.Capping is not important for this scheme.

In the second and third schemes, position is defined by the total massof reporter reached at the stage in synthesis when the residue is addedto the analyte. In this case it is important to terminate part of theextension of the tag by capping a portion of the molecules. The secondand third schemes differ from each other in the way the reporters areadded. In the second they are in the extension agents; in the third theyare in the caps.

FIG. 2. Three types of molecule-specific tags.

A. Illustrates tags made of reporters (E) that specify both position(subscript) and identity (superscript) of the groups in the analyte(U-Z). Such a set could comprise a series of aliphatic chains ofincreasing formula weight to specify position: for example, methylenefor position 1, ethylene for 2, propylene for 3 etc. These could bedifferentiated into group-specific types by different isotopiccompositions of carbon and hydrogen: for example, there are sixdifferent isotopic compositions of CH₂, as shown in Table 1 above. Fourof these differ by one mass unit and should be readily distinguished bymass spectrometry. Other ways of differential labelling can beenvisaged. For example, either position or group could be marked byreporter groups with different charges. Such groups can be separated andrecognised by a number of methods including mass spectrometry.

B. Shows tags made by partial synthesis, such that any structure of theanalyte is attached to a series of tags; the first member of the serieshas a reporter group specific for the first group of the analyte; thesecond has the first reporter plus a second reporter specific for thesecond group of the analyte and so on. Such a series can readily be madeby using two kinds of precursor for extending the tag: one which isprotected by a reversible blocking group and one which prevents furtherextension. For example, a mixture of RX and P-(CH₂)_(n) X, where R is annon-reactive aliphatic group such as methyl or ethyl, P is a reversibleprotecting group and X is an activated residue that can react with thegroup protected by P. Those molecules which have been "capped" by thenon-reactive aliphatic group will not take part in the next round ofdeprotection and extension.

In B the group-specific information is contained in the residues used toextend the synthesis. As in A, the information could be provided usingmass isotopes. For example, every addition of a CH₂ residue labelledwith the isotopes of C and H to P-(CH₂)_(n) X, adds further sites thatcan provide different mass to the reporter. The masses of the (CH₂)_(n)range from 14n to 17n and there are 4+3(n-1) different masses in therange. Thus for the ethylene group there are seven distinct masses inthe range 28 to 34, and for the propylene group, ten in the range 42 to51.

C. Shows how the group-specific information can be added in a differentway; in this case it is contained in the chain terminator, the "cap" inexample B. Again, different masses could be provided by labelling analiphatic residue. Positional information is provided by the length ofthe extension at which the terminator was added. Suppose that E is(CH₂)₂ -O, and the terminators are isotopically labelled methyl groupswith formula weights from 15 to 19. Each extension will add 44 massunits to the reporter. The mass range for the shortest reporter would befrom 44+15=59 to 44+19=63. The range for the second position would befrom 88+15=103 to 88+19=107, and so on to the sixth where the range isfrom 284+15=299 to 284+19=303. There is no overlap in this range, and itcan be seen that the number of reporters and the range could be extendedby using terminators and extensions with more atoms.

LITERATURE CITED

1. Brenner, S. and Lerner, R. A. (1992). Encoded combinatorialchemistry. Proc. Natl. Acad. Sci. USA 89: 5381-5383

2. Drmanac, R., Labat, I., Brukner, I., and Crkvenjakov, R. (1989).Sequencing of megabase plus DNA by hybridization: Theory of the method.Genomics 4: 114-128.

3. Pillai, V. N. R. (1980). Photoremovable protecting groups in organicchemistry. Synthesis 39: 1-26

4. Hoheisel, J. D., Maier, E., Mott, R., McCarthy, L., Grigoriev, A. V.,Schalkwyk, L. C., Nitzetic, D., Francis, F. and Lehrach, H. (1993) Highresolution cosmid and P1 maps spanning the 14 Mbp genome of the fissionyeast Schizosaccharomyces pombe. Cell 73: 109-120.

5. Khrapko, K. R., Lysov, Yu. P., Khorlyn, A. A., Shick, V. V.,Florentiev, V. L., and Mirzabekov. (1989). An oligonucleotidehybridization approach to DNA sequencing. FEBS Lett. 256: 118-122.

6. Patchornik, A., Amit, B. and Woodward, R. B. (1970). Photosensitiveprotecting groups. J. AMER. Chem. Soc. 92: 21: 6333-6335.

7. Ross, M. T., Hoheisel, J. D., Monaco, A. P., Larin, Z., Zehetner, G.,and Lehrach, H. (1992) High density gridded Yac filters; their potentialas genome mapping tools. In "Techniques for the analysis of complexgenomes." Anand, R. ed. (Academic Press) 137-154.

8. Southern, E. M. (1988). Analyzing Polynucleotide Sequences.International Patent Application PCT GB 89/00460.

9. Southern, E. M., Maskos, U. and Elder, J. K. (1992). Analysis ofNucleic Acid Sequences by Hybridization to Arrays of Oligonucleotides:Evaluation using Experimental Models. Genomics 12: 1008-1017.

10. de Vries, M. S., Elloway, D. J., Wendl, R. H., and Hunziker, H. E.(1992). Photoionisation mass spectrometer with a microscope laserdesorption source, Rev. Sci. Instrum. 63(6): 3321-3325.

11. Zubkov, A. M., and Mikhailov, V. G. (1979). Repetitions of s-tuplesin a sequence of independent trials. Theory Prob. Appl. 24, 269-282.

12. Cozzarelli, N. R., Melechen, N. E., Jovin, T. M. and Kornberg, A.(1967). BBRC, 28, 578-586.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATCAAGTCAGAAAAATATATA21                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTGACTTGAT10                                                                  __________________________________________________________________________

We claim:
 1. A method of sequencing a target nucleic acid, which methodcomprises the steps of:a) providing an oligonucleotide immobilized on asupport, b) hybridizing the target nucleic acid with the immobilizedoligonucleotide, c) incubating the hybrid from b) with a librarycomprising a solution of reagents, each of said reagents comprising1) ananalyte moiety comprising at least two analyte residues, and linked to2) a tag moiety comprising one or more reporter groups suitable fordetection by mass spectrometry, excluding oligonucleotides. wherein areporter group designates an analyte residue, and the reporter group ateach position of the tag moiety is chosen to designate an analyteresidue at a defined position of the analyte moiety and wherein thelibrary consists of 4^(n) reagents, each of said reagents comprising adifferent analyte moiety which is a different oligonucleotide chain of nnucleotides, so that an oligonucleotide chain of a first reagent of thelibrary becomes hybridized to the target nucleic acid adjacent theimmobilized oligonucleotide, d) ligating the adjacent oligonucleotides,thus forming a ligated first reagent, e) removing other non-ligatedreagents, and f) recovering and analyzing the tag moiety of the ligatedfirst reagent as an indication of the sequence of a first part of thetarget nucleic acid wherein the said target nucleic acid and ligatedfirst reagent remain hybridized.
 2. The method as claimed in claim 1,comprising the additional steps ofci) incubating the hybrid from f) withthe library comprising a solution of reagents, each of said reagentscomprising1) an analyte moiety comprising at least two analyte residues,and linked to 2) a tag moiety comprising one or more reporter groupssuitable for detection by mass spectrometry, excluding oligonucleotides,wherein a reporter group designates an analyte residue, and the reportergroup at each position of the tag moiety is chosen to designate ananalyte residue at a defined position of the analyte moiety and whereinthe library consists of 4^(n) reagents, each of said reagents comprisinga different analyte moiety which is a different oligonucleotide chain ofn nucleotides, so that an oligonucleotide chain of a second reagent ofthe library becomes hybridized to the target nucleic acid adjacent theoligonucleotide chain of the first reagent, di) ligating the adjacentoligonucleotides, thus forming a ligated second reagent, ei) removingother non-ligated reagents, and fi) recovering and analyzing the tagmoiety of the ligated second reagent as an indication of the sequence ofa second part of the target nucleic acid.
 3. The method as claimed inclaim 1 or claim 2, wherein: in step a) the oligonucleotide isimmobilized on the ends of a series of pins as the support; in step b)an individual clone of target DNA is hybridized to the oligonucleotideimmobilized on each individual pin; in steps c) and d) there are formeda series of ligated reagents, with different pins carrying differentligated reagents; and in step f) the tag moiety of each ligated reagentis recovered and analyzed as an indication of the sequence of a part ofthe target DNA.
 4. The method as claimed in claim 1 or claim 2, wherein:in step b) each individual clone of target DNA is hybridized to theoligonucleotide immobilized at an individual spaced location of thesupport; in steps c) and d) there are provided a series of ligatedreagents with different spaced locations of the support carryingdifferent ligated reagents; and in step f) the tag moiety of eachligated reagent is recovered and analyzed as an indication of thesequence of a part of the target DNA.
 5. A method as claimed in claim 1or claim 2, wherein the method comprises the steps of:a) providing anarray of oligonucleotides immobilized at spaced locations on a support,an oligonucleotide at one location being different from oligonucleotidesat other locations, b) incubating the target nucleic acid with the arrayof immobilized oligonucleotides, so as to form hybrids at one or morespaced locations on the support, c) incubating the hybrids from b) witha library comprising a solution of reagents, each of said reagentscomprising1) an analyte moiety comprising at least two analyte residues,and linked to 2) a tag moiety comprising one or more reporter groupssuitable for detection by mass spectrometry, excluding oligonucleotides.wherein a reporter group designates an analyte residue, and the reportergroup at each position of the tag moiety is chosen to designate ananalyte residue at a defined position of the analyte moiety and whereinthe library consists of 4^(n) reagents, each of said reagents comprisinga different analyte moiety which is a different oligonucleotide chain ofn nucleotides, so that an oligonucleotide chain of a reagent of thelibrary becomes hybridized to the target nucleic acid adjacent eachimmobilized oligonucleotide, d) ligating adjacent oligonucleotides, thusforming ligated reagents at the one or more spaced locations on thesupport, e) removing other non-ligated reagents, and f) recovering andanalyzing the tag moiety of each ligated reagent as an indication of thesequence of a part of the target nucleic acid.
 6. A method as claimed inclaim 5, wherein the sequence is known of the oligonucleotideimmobilized by a covalent bond at each spaced location on the support.7. A library of reagents, wherein each reagent comprisesa) an analytemoiety comprising at least two analyte residues, and linked to b) a tagmoiety comprising one or more reporter groups suitable for detection bymass spectrometry, excluding oligonucleotides, wherein a reporter groupdesignates an analyte residue, and the reporter group at each positionof the tag moiety is chosen to designate an analyte residue at a definedposition of the analyte moiety, wherein the library consists of 4^(n)reagents each comprising a different analyte moiety which is a differentoligonucleotide chain of n nucleotides.
 8. The library as claimed inclaim 7, wherein the reagents are mixed together in solution.
 9. Amethod of analyzing a target DNA, which method comprises the steps of:i)providing the target DNA immobilized on a support, ii) incubating theimmobilized target DNA from i) with a plurality of reagents where eachreagent comprises a) an analyte moiety which is an oligonucleotide chaincomprising at least two nucleotide residues, and linked to b) a tagmoiety comprising one or more reporter groups suitable for detection bymass spectrometry, excluding oligonucleotides, wherein a reporter groupdesignates an analyte residue, and the reporter group at each positionof the tag moiety is chosen to designate an analyte residue at a definedposition of the analyte moiety, so that the oligonucleotide chains ofdifferent reagents become hybridized to the target DNA on the support,iii) removing non-hybridized reagents, and iv) recovering and analyzingthe tag moiety of each reagent as an indication of the sequence of apart of the target DNA wherein the said target DNA and any hybridizedreagents remain hybridized.
 10. A method as claimed in claim 9,comprising the additional steps of:iia) incubating the hybrid from iv)with a library of reagents comprising a solution of reagents, each ofsaid reagents comprising1) an analyte moiety comprising at least twoanalyte residues, and linked to 2) a tag moiety comprising one or morereporter groups suitable for detection by mass spectrometry, excludingoligonucleotides, wherein a reporter group designates an analyteresidue, and the reporter group at each position of the tag moiety ischosen to designate an analyte residue at a defined position of theanalyte moiety, and wherein the library consists of 4^(n) reagents, eachof said reagents comprising a different analyte moiety which is adifferent oligonucleotide chain of n nucleotides, so thatoligonucleotide chains of different reagents become hybridized to thetarget DNA, iiia) ligating adjacent oligonucleotides hybridized to thetarget DNA and removing non-ligated reagents; and iva) recovering andanalyzing the tag moiety of each ligated reagent as an indication of thesequence of part to the target DNA.